The Mechanisms Of Insecticide Resistance Induced By Plant Secondary Metabolite Flavone In Spodoptera Litura(Fabricius)

Flavonoids are important defensive secondary metabolites of plants,which play an important role in plant resistance to herbivorous insects,pathogenic bacteria infection and weed control.Spodoptera litura(Fabricius)is a kind of explosive and omnivorous pest,its insecticide resistance is an important problem in agricultural production.Cytochrome P450 enzyme system(P450),Glutathione S-transferase(GST)and carboxylesterase(CarE)are the main detoxification enzymes for insect resistance to toxic substances(including plant secondary metabolites,insecticides and heavy metals).Flavone is a representative substance of flavonoids,some studies have shown that the GST activity of S.litura was enhanced after ingestion of flavone,but the effect of S.litura insecticide resistance after exposed to flavone was not reported so far.In this research,the resistance of S.litura to insecticides and the mechanism were studied by adding different concentrations of flavone in artificial diet.The main findings are as follows:1.The resistance of S.litura exposed to chlorpyrifos,imidacloprid,fipronil and λ-cyhalothrin diet increased significantly after feeding on artificial diet contained 0.25,0.5,0.75 mg/g flavone for 48 h.Compared with the control group,the resistance of S.litura to chlorpyrifos increased by 98.69%,290.78%and 264.24%respectively.The tolerance to imidacloprid increased by 21 1.44%,244.73%and 274.06%.The resistance of S.litura after dropped chlorpyrifos and β-cypermethrin was also improved significantly by feeding on the artificial diet contained flavone.Compared with the control group,the P450 enzyme activity in midgut and fat body of S.litura were increased by 34.4%-50.9%and the CarE enzyme activity were increased by 38.1%-239.5%after feeding on artificial diet contained different concentrations of flavone.GST enzyme activity in fat body of S.litura was increased by 48%-74%.These results suggest that the insecticide resistance of S.litura was improved after exposed to flavone,and P450,CarE and GST detoxification enzymes may play an important role.2.qRT-PCR results showed that multiple P450 and CarE genes of S.litura were induced after larvae exposed to flavone.Two P450 genes CYP6AE43 and CYP6AE44,two CarE genes CCE006b and CCE016d were selected for functional verification.The full-length sequence of the genes was cloned by using 5’ and 3’ RACE,the conserved domains of the genes were identified,CYP6AE43 and CYP6AE44 were belong to the P450 super family,CCE006b and CCE016d were belong to the Abhydrolase super family.The P450 and CarE proteins have conserved C-terminal heme binding region of the P450 family and CarE-specific serine active site,respectively.The protein phylogenetic tree was constructed to find the evolutionary relationship with other species.It was found that the two P450 genes were relatively conservative in evolution,but the two CarE genes were far related to other species.qRT-PCR showed that after exposed to flavone,CYP6AE43 was up-regulated by more than 10 times in midgut of S.litura,CYP6AE44 was up-regulated by more than 2 times in fat body,CCE006b was up-regulated by more than 2 times in midgut and CCE016d was up-regulated by about 1 times in fat body.Tissue distribution and development patterns suggest that CYP6AE43,CYP6AE44,CCE006b,CCE016d were highly expressed in larvae stage of S.litura,CYP6AE43 and CCE006b were highly expressed in midgut,CYP6AE44 and CCE016d were highly expressed in fat body.These genes were highly expressed in midgut or fat body and significantly up-regulated after larvae exposed to flavone,the expression of these genes was positive correlation to the insecticide resistance after flavone treatment,suggesting that these genes may be related to detoxification of S.litura.After RNAi silenced the expression of CYP6AE43,CCE006b,CCE016d genes,the sensitivity of S.litura to chlorpyrifos was significantly increased,which indicated that these three detoxification enzyme genes had significant influence on the insecticide resistance of S.litura,and these genes may be involved in the flavone-induced insecticide resistance of S.litura.But after RNAi CYP6AE44,the insecticide resistance of S.litura was not change.This study shows that flavone can induce S.litura to have insecticide resistance to many kinds of insecticide,which may be caused by the upregulation of CYP6AE43,CCE006b,CCE016d genes expression and the enhancement of P450,GST and CarE detoxification enzyme activities induced by flavone.After RNAi silenced the expression of CYP6AE43,CCE006b,CCE016d genes,the resistance of S.littura to chlorpyrifos was significantly reduced,providing potential molecular targets for the future development of new insecticides by using bioengineering technology.